Msc1 acts through histone H2A.Z to promote chromosome stability in Schizosaccharomyces pombe

Abstract

As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components. Multicopy expression of msc1 robustly suppresses a temperature-sensitive mutant (cnp1-1) in the centromere-specific histone H3 variant CENP-A, and localization of CENP-A to the centromere is compromised in msc1 null cells. We present several lines of evidence to suggest that Msc1 carries out its function through the histone H2A variant H2A.Z, encoded by pht1 in fission yeast. Like an msc1 mutant, a pht1 mutant also exhibits chromosome instability and genetic interactions with kinetochore mutants. Suppression of cnp1-1 by multicopy msc1 requires pht1. Likewise, suppression of the DNA damage sensitivity of a chk1 mutant by multicopy msc1 also requires pht1. We present the first genetic evidence that histone H2A.Z may participate in centromere function in fission yeast and propose that Msc1 acts through H2A.Z to promote chromosome stability and cell survival following DNA damage.

Figure 1.—

Msc1 is required for chromosome stability. (A) Strains with a cdc25-22 mutant allele were synchronized in G₂ by incubation at 36.5° and released to permissive temperature. After 90 min, cells were fixed with gluteraldehyde and processed for immunofluorescence with antitubulin antibody (red). Nuclei are stained with DAPI (blue). Portions of the micrographs in boxes are enlarged for better viewing of normal (left) and lagging (right) chromosomes (white arrows). (B) Schematic of chromosome loss assay. Ch16 is a minichromosome carrying the ade6-216 allele that complements in trans the ade6-210 allele (Niwa et al. 1986). Loss of Ch16 results in cells that solely express the ade6-210 allele, which confers a red color to the colonies. From the parent strains that are either msc1⁺ or msc1∷kan^R, single white colonies were grown as described in materials and methods. Equal numbers of cells of the indicated strains were plated on plates with a limiting concentration of adenine.

Figure 2.—

Msc1 exhibits genetic interactions with Mis12 and Mis6. (A and B) The indicated strains were grown at 25° to mid-log phase. Tenfold serial dilutions were spotted on rich media plates and incubated at the indicated temperatures for 3–4 days.

Figure 3.—

An msc1 null is not synthetically sick with cnp1-1, but multicopy msc1 suppresses loss of function of CENP-A. (A) The indicated strains were grown at 25° to mid-log phase. Tenfold serial dilutions were spotted on rich media plates and incubated at the indicated temperatures for 3–4 days. (B and C) The cnp1-1 (B) or cnp1-1 mis6 (C) strain was transformed with the indicated plasmids. Cells were grown at 25° in minimal media to mid-log phase. Tenfold serial dilutions were spotted on minimal media plates and incubated at the indicated temperatures for 3–4 days.

Figure 4.—

CENP-A localization to the inner centromeric region is reduced in the absence of Msc1. Cells with GFP-tagged Cnp1 (CENP-A) with wild-type or a null allele of msc1, or an untagged control, were subjected to chromatin immunoprecipitation with antibody to GFP as described in materials and methods. DNA from the lysates (input) or the immunoprecipitations were subject to PCR with primers specific to the central (cnt), inner (imr), and outer (otr) regions of the fission yeast centromere on chromosome I. Enrichment of imr1 and cnt1 was assessed compared to otr1 in immunoprecipitates relative to input DNA. This experiment was performed three times with similar results.

Figure 5.—

A pht1 null mutant exhibits genetic interactions similar to msc1. (A and B) The indicated strains were grown at 25° to mid-log phase. Tenfold serial dilutions were spotted on rich media plates and incubated at the indicated temperatures for 3–4 days. (C) The cnp1-1 or cnp1-1 pht1Δ strain was transformed with the indicated plasmids. Cells were grown at 25° in minimal media to mid-log phase. Tenfold serial dilutions were spotted on minimal media plates and incubated at the indicated temperatures for 3–4 days.

Figure 6.—

The ability of multicopy Msc1 to restore survival of a chk1Δ strain requires histone H2A.Z. The indicated strains transformed with the indicated plasmids were grown in minimal media to mid-log phase at 25°. Serial dilutions (10-fold) were spotted on yeast nitrogen media plates lacking leucine and incubated at the indicated temperatures.

Figure 7.—

The ability of multicopy Msc1 to restore survival of a chk1Δ strain requires Mad2 function, although Msc1 is not required directly for the spindle checkpoint. (A) The indicated strains transformed with the indicated plasmids were grown in minimal media to mid-log phase at 25°. Serial dilutions (10-fold) were spotted on yeast nitrogen media plates lacking leucine and incubated at the indicated temperatures. (B) Cells were blocked in G₂ by incubation at 36.5° and then released at permissive temperature in the presence of TBZ (50 μg/ml). At 20-min intervals cells were fixed with gluteraldehyde and processed for immunofluorescence with antitubulin antibody and stained with DAPI. For wild-type and msc1Δ cells, the 100-min time point is shown. For mad2Δ cells, the 60-min time point is shown.